Polypeptide or derivative thereof, preparation method therefor, and use thereof in promoting skin moisturization

Abstract

A moisturizing polypeptide or a derivative thereof, wherein the moisturizing polypeptide is selected from 3H-12 or a polypeptide fragment having an amino acid sequence homology of 70% or higher with the amino acid sequence of 3H-12, the amino acid sequence of the polypeptide 3H-12 being shown in SEQ ID NO.1, and the amino acid sequence of the polypeptide fragment being shown in SEQ ID NO.2-5. The polypeptide derivative is a product obtained by conventional modification on the amino acid side chain, amino terminus, or carboxyl terminus of the moisturizing polypeptide, by attachment of a tag molecule, or by isotopic labeling modification. The moisturizing polypeptide and the derivative thereof have a good effect on skin moisturization, and can up-regulate the expression of aquaporin and cell proliferation of keratinocytes, significantly increasing the moisture content of the skin stratum corneum, and ameliorating the problem of sensitive skin caused by skin dehydration.

Description

A polypeptide or its derivative, its preparation method, and its application in promoting skin hydration. Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a polypeptide 3H-12 that promotes skin hydration, its derivatives, and their applications. Background Technology

[0002] As the largest organ in the human body, the skin covers internal organs and its main function is to protect the body from harmful external stimuli such as microorganisms, viruses, and radiation. In addition, the stratum corneum of the skin has a water-retention function, containing natural moisturizing factors (NMF) and a lipid barrier (such as ceramides, fatty acids, and cholesterol), effectively reducing water evaporation. Moisturizing is fundamental to repairing the skin barrier; ingredients with good moisturizing effects can prevent moisture loss and help restore the skin's barrier function, such as hyaluronic acid, glycerin, and triglycerides.

[0003] Peptides play a crucial role in human life activities, acting as messengers for physiological and biochemical reactions and maintaining the stability of these processes. They are hailed as one of the greatest discoveries in human history. Due to their high activity and diversity, peptides are a hot topic in research and development in the biological, medical, and pharmaceutical fields, and have been widely applied in pharmaceuticals, cosmetics, and health foods. Cosmetic peptides are a very important class of ingredients in modern skincare. They improve skin health, slow aging, repair damage, moisturize, and brighten the skin through various mechanisms. They can penetrate deep into the skin layers to exert lasting and significant effects, suitable for various skincare needs such as anti-aging, repair, moisturizing, and whitening. Cosmetic peptides are highly effective and low-irritant, and have been widely used in people with various skin conditions. However, current cosmetic peptides have limited selectivity and may not meet the body's moisturizing needs, resulting in poor moisturizing effects and exaggerated claims. Currently, there is a lack of truly effective and safe peptide products on the market for customers to choose from. Summary of the Invention

[0004] In view of the problems existing in the prior art, the purpose of this invention is to design and provide a polypeptide that promotes skin hydration and its application, providing a new approach to the addition of polypeptides to cosmetics.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] On the one hand, the present invention provides a polypeptide or its derivative having a moisturizing function, wherein the polypeptide having a moisturizing function is 3H-12, or a polypeptide fragment having more than 70% homology with 3H-12.

[0007] The amino acid sequence of 3H-12 is shown in SEQ ID NO.1;

[0008] The amino acid sequence of the polypeptide fragment is shown in any one of SEQ ID NO.2-5.

[0009] The aforementioned polypeptide or its derivative having moisturizing function, wherein the polypeptide derivative is a product obtained by conventional modification of the amino acid side chain groups, amino terminus or carboxyl terminus of the polypeptide, a product obtained by attaching a tag, or a product obtained by isotope labeling modification.

[0010] The aforementioned polypeptide or its derivative with moisturizing function is conventionally modified by isotope labeling, fluorescent group modification, phosphorylation modification, disulfide bond-based cyclization modification, biotin labeling modification, photosensitizer modification (which can be used to prepare photosensitizing agents), azide modification (which can be used for secondary linkage reactions), PEG modification (which can be used to prepare drug carriers), methylation modification, fluorescence quencher group modification, protein coupling modification, small molecule compound modification, aminoation modification, amidation modification, hydroxylation modification, carboxylation modification, carbonylation modification, alkylation modification, acetylation modification, esterification modification, nitration modification, or glycosylation modification.

[0011] The aforementioned polypeptide or its derivative having moisturizing function, wherein the polypeptide derivative is a product obtained by acetylation modification at the amino terminus of the polypeptide, a product obtained by conventional modification at the carboxyl terminus of the polypeptide, or a product obtained by modification at the R group of the amino acid side chain of the polypeptide.

[0012] The aforementioned polypeptide or its derivative with moisturizing function, wherein the fluorescent group modification includes AMCA, FITC, Rhodamine, Cy3, Cy5, Cy5.5, Cy7, AIE, or ICG; this modification can be used for fluorescence detection.

[0013] The phosphorylation modification is p-Ser, p-Thr, or p-Tyr modification;

[0014] The glycosylation modification is a Ser, Asn, Thr, or Tyr modification;

[0015] The nitration modification is a Tyr modification;

[0016] The biotin in the biotin labeling modification is D-biotin, biotinyl hydrazide, photosensitive biotin, or biotin-dUTP;

[0017] The isotopes used in the isotope labeling modification are 13C, 14C, 14N, 15N, 2H, 3H, 18O, 32P, 32S, 34S, 35S, 36S, 35Cl, 37Cl, 125I, or 131I.

[0018] Secondly, the present invention provides a method for preparing a polypeptide or its derivative having a moisturizing function, wherein the polypeptide having a moisturizing function is obtained by extraction and separation, biosynthesis or chemical synthesis.

[0019] A method for preparing a polypeptide or its derivative with moisturizing function, wherein the method for preparing the polypeptide with moisturizing function specifically comprises: coupling the protecting amino acids to the resin sequentially from the C-terminus to the N-terminus according to the amino acid sequence of the polypeptide; obtaining the crude peptide precursor by lysis with a lysis buffer; and then obtaining the moisturizing polypeptide by high performance liquid chromatography purification, salt transfer, and freeze drying.

[0020] Preferably, the pyrolysis solution is a pyrolysis solution containing trifluoroacetic acid;

[0021] The preparation method of the polypeptide derivative is as follows: the polypeptide is conventionally modified or a tag is attached to obtain the polypeptide derivative.

[0022] The elution conditions for peak collection were as follows: mobile phase A: 20 mM ammonium bicarbonate solution, mobile phase B: acetonitrile, flow rate: 400 ml / min, wavelength: 230 nm, elution gradient (B): 5% (5 min), 12% (60 min), 22%, and the target peak was collected in segments.

[0023] In the neutralization reaction for preparing peptides, sodium hydroxide, an alkaline reagent, is used to adjust the solubility of the peptides, and the resulting peptides are sodium salt peptides.

[0024] The aforementioned moisturizing polypeptide has a pH value of 7-8, classifying it as a neutral polypeptide.

[0025] The use of the aforementioned moisturizing polypeptide or its derivative in the preparation of skin-moisturizing cosmetics, medical aesthetic products, pharmaceuticals, or additives.

[0026] Thirdly, the present invention provides an preparation for promoting skin hydration, comprising a therapeutically effective amount of a polypeptide or its derivative having a moisturizing function as described in claim 1 and a variety of pharmaceutically acceptable carriers;

[0027] The preparation is a cosmetic, medical aesthetic product, pharmaceutical, or additive.

[0028] The aforementioned preparation for promoting skin hydration, wherein the carrier is selected from one or more of diluents, excipients, fillers, binders, humectants, disintegrants, absorption promoters, adsorbent carriers, surfactants, or lubricants.

[0029] The dosage form of the preparation is selected from one or more of the following: tablets, granules, capsules, oral liquids, inhalation liquids, topical liquids, sprays, drops, microneedles, or injections.

[0030] The formulation is administered orally or via local contact.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] Moisturizing peptides can upregulate the expression of aquaporins and cell proliferation in keratinocytes, significantly increase the moisture content of the stratum corneum, and improve sensitive skin problems caused by dehydration. This provides new ideas for the addition of beauty peptide raw materials to cosmetics and offers new solutions for sensitive skin problems. Attached Figure Description

[0033] Figure 1 shows the polypeptide content detected by HPLC.

[0034] Figure 2 shows the cell viability assay using the MTT assay.

[0035] Figure 3 is a summary of the AQP3 immunofluorescence staining results;

[0036] Figure 4 is a bar chart of the relative integrated optical density (IOD) values ​​of AQP3. Detailed Implementation

[0037] To better understand the content of this invention, the following embodiments describe specific aspects of the invention to explain the invention and provide a description of the methods of the invention for those skilled in the art, but the scope of protection of this invention is not limited to the following embodiments.

[0038] Example 1: Preparation of moisturizing peptide 3H-12

[0039] (1) Using Wang Resin as the starting resin, DMF was swollen;

[0040] (2) Add amino acid Fmoc-Leu-OH / HOBt / DIC / DMAP=2 / 2.2 / 2.6 / 0.2 for coupling, and after a reaction time of about 6 hours, Fmoc-Leu-Wang Resin is obtained;

[0041] (3) The amino protecting group (Fmoc) was removed by 20% PIPE / DMF solvent to obtain NH2-Leu-Wang Resin (K test positive);

[0042] (4) Then, it was coupled with the amino acid Fmoc-Ser(tBu)-OH / HOBt / DIC=2.0 / 2.2 / 2.6 coupling system. The temperature was controlled at 25±2℃ and the reaction time was about 2 hours (K test was negative) to obtain Fmoc-Ser(tBu)-Leu-Wang Resin;

[0043] (5) Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Thr(tBu)-OH, and Fmoc-Ala-OH were gradually coupled by repeated coupling process to obtain 3H-12 peptide resin;

[0044] (6) Shrink the resin with MeOH and DCM, and obtain the weight of peptide resin after drying;

[0045] (7) Then add 10 times the volume of TFA / Tis / H2O = 92.5:2.5:2.5 (volume ratio) pyrolysis solution and pyrolyze for 2 hours. Concentrate to remove 1 / 3 to 1 / 2 of the volume of pyrolysis solution (concentration conditions: temperature ≤32℃, vacuum degree ≤-0.08Mpa);

[0046] (8) The product was precipitated with 6.5 times its volume of isopropyl ether, washed, and centrifuged to obtain crude C2773. After purification by reversed-phase HPLC, the final product was obtained by salting and yielding polypeptide 3H-12.

[0047] The results are shown in Figure 1. The purity of peptide 3H-12 was determined to be 96.65% by HPLC.

[0048] Example 2: Cytotoxicity assay

[0049] (1) Cell seeding: at 1×10 4 HaCaT cells were seeded into 96-well plates at a seeding density of cells / well and incubated overnight in an incubator (37°C, 5% CO2).

[0050] (2) Experimental grouping: The experiment was set up with a zeroing group, a control group, a positive control group and a sample group. In the sample group, each sample was set with 8 concentration gradients, and 3 replicate wells were set under each concentration gradient. The specific samples and concentrations are shown in Table 1 below;

[0051] Table 1 Sample and Concentration Setting Table

[0052] (3) Solution preparation: Prepare peptide dilution solutions of different concentrations using 10% PBS according to the sample concentration setting table;

[0053] (4) Drug administration: Drug administration was performed when the cell seeding rate in the 96-well plate reached 40%-60%. 200 μL of culture medium containing 10% PBS was added to each well of the control group; 200 μL of culture medium containing 10% DMSO was added to each well of the positive control group; 200 μL of culture medium containing the corresponding concentration of the sample was added to each well of the sample group; no cells were seeded in the zeroing group, only 200 μL of cell culture medium was added. After drug administration, the 96-well plate was placed in an incubator (37℃, 5% CO2) for culture.

[0054] (5) Detection: After culturing cells for 24 hours, discard the supernatant, add MTT working solution (0.5 mg / mL), and incubate at 37°C in the dark for 4 hours. After incubation, discard the supernatant, add 100 μL of DMSO to each well, and read the OD value at 490 nm.

[0055] (6) Calculate cell viability according to the following formula I.

[0056] The results are shown in Figure 2. A cell viability graph was plotted with the eight selected concentrations of peptide 3H-12 as the x-axis and cell viability as the y-axis. The MTT results showed that sample dodecapeptide-1 did not exhibit keratinocyte cytotoxicity in the concentration range of 0.500% (m / V), indicating that the peptide has good safety.

[0057] Example 3: Aquaporin AQP3 Content Test

[0058] (1) Cell seeding: at 1×10 5 HaCaT cells were seeded into 24-well plates at a seeding density of cells / well and incubated overnight in an incubator (37°C, 5% CO2).

[0059] (2) Solution preparation: Prepare three concentrations of polypeptide 3H-12 dilutions of 0.001% / 0.01% / 0.03% using HaCaT cell culture medium.

[0060] (3) Drug administration: When the cell deposition rate in the 24-well plate reaches 40%-60%, the original cell culture medium is aspirated, and drugs are administered to the groups. For the experimental group, 1.0 mL of sample working solution is added to each well, with three replicates for each concentration of working solution. For the control group, 1.0 mL of cell culture medium is added to each well, with three replicates. The plates are then placed in an incubator (37℃, 5% CO2) and cultured for another 24 hours.

[0061] (4) Sample collection: Discard the supernatant and rinse the cells 3 times with PBS.

[0062] (5) Immunofluorescence staining: Perform routine immunofluorescence staining. The main steps are: fixation, blocking, adding primary antibody, adding secondary antibody, DAPI counterstaining, and then taking pictures using a fluorescence microscope.

[0063] (6) Results analysis: Quantitative analysis of AQP3 fluorescence intensity was performed.

[0064] The results are shown in Figures 3 and 4, where green fluorescence represents AQP3 and blue fluorescence represents the cell nucleus. Aquaporin (AQP3) is a subfamily of the aquaporin family and is the most expressed aquaporin in the skin. It is expressed in the epidermis and basal layer, maintaining the hydration level of the intercellular matrix and intracellular spaces, and plays a key role in skin moisturization. Based on the human immortalized keratinocyte (HaCaT) model, peptide 3H-12 significantly enhanced the fluorescence intensity of aquaporin (AQP3) at concentrations of 0.01% and 0.03% (m / V), indicating that peptide 3H-12 can increase the AQP3 content in keratinocytes and has a moisturizing effect.

Claims

1. A polypeptide or its derivative having moisturizing function, characterized in that, The moisturizing polypeptide is 3H-12, or a polypeptide fragment with more than 70% homology to 3H-12. The amino acid sequence of 3H-12 is shown in SEQ ID NO.1; The amino acid sequence of the polypeptide fragment is shown in any one of SEQ ID NO.2-5.

2. A polypeptide or its derivative having moisturizing function as described in claim 1, characterized in that, The polypeptide derivative is a product obtained by conventional modification of the amino acid side chain groups, amino terminus, or carboxyl terminus of the polypeptide, a product obtained by attaching a tag, or a product obtained by isotope labeling modification.

3. A polypeptide or its derivative having moisturizing function as described in claim 2, characterized in that, The conventional modification methods include fluorescent group modification, phosphorylation modification, disulfide bond-based cyclization modification, biotin labeling modification, photosensitizer modification, azide modification, PEG modification, methylation modification, fluorescence quencher group modification, protein coupling modification, small molecule compound modification, aminoation modification, amidation modification, hydroxylation modification, carboxylation modification, carbonylation modification, alkylation modification, acetylation modification, esterification modification, nitration modification, or glycosylation modification.

4. A polypeptide or its derivative having moisturizing function as described in claim 2, characterized in that, The polypeptide derivative is a product obtained by acetylation modification at the amino terminus of the polypeptide, a product obtained by conventional modification at the carboxyl terminus of the polypeptide, or a product obtained by modification at the R group of the amino acid side chain of the polypeptide.

5. A polypeptide or its derivative having moisturizing function as described in claim 3, characterized in that, The fluorescent group used in the fluorescent group modification is AMCA, FITC, Rhodamine, Cy3, Cy5, Cy5.5, Cy7, AIE, or ICG; The phosphorylation modification is p-Ser, p-Thr, or p-Tyr modification; The glycosylation modification is a Ser, Asn, Thr, or Tyr modification; The nitration modification is a Tyr modification; The biotin in the biotin labeling modification is D-biotin, biotinyl hydrazide, photosensitive biotin, or biotin-dUTP; The isotopes used in the isotope labeling modification are 13C, 14C, 14N, 15N, 2H, 3H, 18O, 32P, 32S, 34S, 35S, 36S, 35Cl, 37Cl, 125I, or 131I.

6. The method for preparing a polypeptide or its derivative with moisturizing function as described in claim 1, characterized in that, The moisturizing polypeptides are obtained through extraction, separation, biosynthesis, or chemical synthesis.

7. The preparation method according to claim 6, characterized in that, The preparation method of the aforementioned moisturizing peptide is as follows: the protective amino acids are coupled to the resin sequentially from the C-terminus to the N-terminus according to the amino acid sequence of the peptide, the crude peptide precursor is obtained by lysis with lysis buffer, and then purified by high performance liquid chromatography, converted to salt, and freeze-dried to obtain the moisturizing peptide. Preferably, the pyrolysis solution is a pyrolysis solution containing trifluoroacetic acid; The preparation method of the polypeptide derivative is as follows: the polypeptide is conventionally modified or a tag is attached to obtain the polypeptide derivative.

8. The use of a moisturizing polypeptide or its derivative as described in claim 1 in the preparation of skin-moisturizing cosmetics, medical aesthetic products, pharmaceuticals or additives.

9. A preparation for promoting skin hydration, characterized in that, The product comprises a therapeutically effective amount of the moisturizing polypeptide or its derivatives as described in claim 1, and various pharmaceutically acceptable carriers; The preparation is a cosmetic, medical aesthetic product, pharmaceutical, or additive.

10. A skin moisturizing agent as described in claim 8, characterized in that, The carrier is selected from one or more of the following: diluent, excipient, filler, binder, wetting agent, disintegrant, absorption promoter, adsorbent carrier, surfactant, or lubricant; The dosage form of the preparation is selected from one or more of the following: tablets, granules, capsules, oral liquids, inhalation liquids, topical liquids, sprays, drops, microneedles, or injections. The formulation is administered orally or via local contact.

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